Altered DNA conformations detected by mung bean nuclease occur in promoter and terminator regions of supercoiled pBR322 DNA

Sheflin, Lowell G.; Kowalski, David

doi:10.1093/nar/13.17.6137

Cited by 81 publications

(69 citation statements)

References 68 publications

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“…When a plasmid DNA containing ARSJ in pBR322-derived vector was digested with mung bean nuclease in the absence of HSSB, the cleavage signal was strongest in the terminator region of the ampicillin resistance gene in the vector DNA and weak signals were detected in the ARS1 region. These results are in good agreement with those of Sheflin and Kowalski (43), who found that when pBR322 DNA was digested with mung bean nuclease under low ionic conditions, the terminator region of the ampicillin resistance gene was preferentially cleaved. The unwinding element in the terminator region of the ampicillin resistance gene is 84 bp long and highly A+T rich.…”

Section: Discussionsupporting

confidence: 92%

“…On the digestion of negatively supercoiled vector DNA (pBR322AEP), a pair of new bands, named band 5, appeared; these bands mapped at the 3' end of the RNA1 gene ( Fig. 4B and data not presented) (43), suggesting that the most sensitive site can be altered by the DNA context of the plasmid. In the presence of HSSB, the DNA in region 1, in addition to the DNA in region 5, was digested with the nuclease.…”

Section: Resultsmentioning

confidence: 98%

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Single-stranded-DNA-binding protein-dependent DNA unwinding of the yeast ARS1 region.

Matsumoto

Ishimi

1994

Mol. Cell. Biol.

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DNA unwinding of autonomously replicating sequence 1 (ARSI) from the yeast Saccharomyces cerevisuie was investigated. When a negatively supercoiled plasmid DNA containing ARS) was digested with single-strandspecific mung bean nuclease, a discrete region in the vector DNA was preferentially digested. The origin of DNA replication is defined as the site where replication initiates. In bacteria such as Escherichia coli and Bacillus subtilis, replication initiates from a single chromosomal origin (for a review, see reference 5). In the yeast Saccharomyces cerevisiae, autonomously replicating sequences (ARS) are well-defined chromosomal origins that function in plasmids (40,45,47). ARS elements confer upon their plasmid the ability to replicate in yeast cells, allowing the plasmid to transform the yeast cells at a high frequency. A sequence comparison among identified ARS elements revealed that the ARS core consensus sequence consists of 11 bp of 5'-(T/ A)TrrA(C/T)(G/A)TT1'(T/A)-3', which is essential for origin function (8,52). Point mutations in the core consensus sequence cause the loss of ARS activity (10, 24, 52). A flanking region located adjacent to the 3' side of the T-rich strand of the consensus sequence is also required for efficient replication. The 3'-flanking region is A+T rich and contains several sequences that almost match the core consensus (41). Deletions in the flanking region lead to increases in the rate of plasmid loss, whereas point mutations in the similar core consensus sequences have little effect onARS function (24, 41), suggesting that a feature(s) other than the sequence is important for the activity.ARS1 is derived from a replication origin of chromosome IV (6). Analysis of the transformation activity of deletion mutants with mutations in ARS1 has shown that ARS1 consists of domains A, B. and C (Fig. 1) (10, 44). The A domain is a core consensus sequence that is fully matched. The 3'-flanking B domain of about 100 bp contains three 9-of-11 matched consensus sequences and a binding site for protein ABF1 (13 (32) have shown, by using linker substitution mutants, that three distinct regions (B1, B2, and B3) in the B domain are required for ARS function. The C domain of about 80 bp is unique in ARS1 and is located 200 bp away from the A domain on the opposite side from the B domain. When the C domain is deleted, the rate of plasmid loss increases two-to threefold (27,40,46). The initial step of DNA replication is the DNA unwinding at or near the origin, which provides an entry site for replication enzymes. Initiator proteins, which have been identified in bacteria and eukaryotic virus systems, facilitate the unwinding near the origin by binding to specific sequences (4, 5). Kowalski and coworkers (29,50,51) showed the DNA-unwinding property of S. cerevisiae and E. coli replication origins by using single-strand-specific nucleases as probes and proposed that the ease of DNA unwinding is one of the determinants of DNA replication origins. They showed that the 3'-flanking region of the ARS co...

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Section: Discussionsupporting

confidence: 92%

Section: Resultsmentioning

confidence: 98%

Single-stranded-DNA-binding protein-dependent DNA unwinding of the yeast ARS1 region.

Matsumoto

Ishimi

1994

Mol. Cell. Biol.

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“…The removal of terminators should also identify clones in which the terminators had been increasing stability in some manner other than the prevention of read-through transcription. For example, by changing the topology of the plasmid, the hairpin structures of transcription terminators might reduce conformational changes associated with AT-rich DNA (34), which can cause deletions (14). However, we observed that when the terminators were removed, a large number of stable clones containing the expected fragment was obtained for each of the plasmids listed in Table 1, except XPL.…”

Section: Methodsmentioning

confidence: 84%

Analysis of Streptococcus pneumoniae sequences cloned into Escherichia coli: effect of promoter strength and transcription terminators

Dillard

Yother

1991

J Bacteriol

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Difficulties encountered in the cloning of DNA from Streptococcus pneumoniae and other AT-rich organisms into ColEl-type Escherichia coli vectors have been proposed to be due to the presence of a large number of strong promoter-acting sequences in the donor DNA. The use of transcription terminators has been advocated as a means of reducing instability resulting from disruption of plasmid replication caused by strong promoters. However, neither the existence of promoter-acting sequences of sufficient strength and number to explain the reported cloning difficulties nor their role as a source of instability has been proven. As a direct test of the "strong promoter" hypothesis, we cloned random fragments from S. pneumoniae into an E. coli vector containing transcription terminators, identified strong promoter-acting sequences, and subsequently removed the transcription terminators. We observed that terminator removal resulted in reduced copy numbers for the strongest promoter-acting sequences but not in reduced promoter strengths or altered plasmid stabilities. Our results indicate that promoters strong enough to require transcription terminators for plasmid stability are probably rare in S. pneumoniae DNA.Attempts to clone DNA from Streptococcus pneumoniae into ColEl-type multicopy plasmids of Escherichia coli have often met with significant difficulty (8,23,24,29,32,35). It has been proposed that this difficulty is related either to the production of pneumococcal proteins that are detrimental to E. coli or to the presence of large numbers of random sequences with strong promoter activity that result in instability in E. coli. The former explanation accounts for the inability to clone amiA and maiX in the absence of either mutations in the cloned sequences or reductions in plasmid copy number (23, 35). However, certain S. pneumoniae sequences, e.g., those containing the genes com, hexA, hexB, and recP, have proven unstable in ColEl-type multicopy vectors in E. coli but have been cloned by utilizing plasmids of gram-positive bacteria in S. pneumoniae or by using ColEl-type multicopy vectors containing transcription termination sequences for cloning in E. coli (8,24,29,32). Vectors containing transcription terminators have been reported to increase the efficiency of shotgun cloning of S. pneumoniae DNA and to increase the number of random, stable clones obtained, compared with an identical vector lacking terminators (9).Because S. pneumoniae DNA is 61% AT (22), the likelihood of cloning random sequences that resemble the E. coli a70 promoter sequence is high (28). To recognize actual promoters, S. pneumoniae RNA polymerase is expected to have a more stringent consensus sequence requirement than does E. coli RNA polymerase (26, 27). Indeed, it has been shown that more strong promoter-acting sequences are obtained with random S. pneumoniae DNA fragments than with random E. coli fragments (9). In addition, we have observed that S. pneumoniae sequences that function as promoters in E. coli do not necessarily do so in S. ...

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“…As described in the previous section, hot spot III in pUC118 was located in a 6-bp spacer sequence flanked by 10-bp inverted repeat sequences (Fig. 3B), which are entirely contained in the transcription terminator for bla (28). To test the possibility that the inverted repeats flanking a hot spot are responsible for preferential transposition of Tn4731, therefore, we introduced substitution mutations within each one of the inverted repeat sequences flanking hot spot III and examined transposition of Tn4731 to the resulting mutant plasmids pTT70 and pTT71 (Fig.…”

Section: Resultsmentioning

confidence: 70%

Preferential transposition of an IS630-associated composite transposon to TA in the 5'-CTAG-3' sequence

Tenzen

Ohtsubo

1991

J Bacteriol

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A composite transposon, Tn4731, associated with IS630 has been shown to transpose preferentially to 5'-TA-3' sequences that are located at two sites in a rho-dependent transcription terminator in plasmid ColEl in Escherichia coli (T. Tenzen, S. Matsutani, and E. Ohtsubo, J. Bacteriol. 172:3830-3836, 1990). Here we demonstrated that Tn4731 preferentially transposes to TA sequences at four sites in plasmid pUC118 and its derivatives: the TA sequence (hot spot I) in the intergenic region of phage M13 within the pUC sequence, the TA sequence (hot spot II) in the XbaI site in multiple cloning sites of the lacZ coding region, the TA sequence (hot spot III) in a spacer region flanked by inverted repeat sequences of a transcription terminator located downstream of the bla gene, and the TA sequence (hot spot IV) in the middle of bla. Transposition of Tn4731 to hot spot III was found not to require the inverted repeats in the terminator. Transposition of Tn4731 to hot spot II, which is located immediately downstream of the lacZ promoter, was not affected by mutations introduced into the promoter. There appear to be no particular sequences important for transposition of Tn4731 around each of the hot spots, except a palindromic sequence, 5'-CTAG-3', that contains the target sequence. Mutations introduced within the CTAG sequence at a hot spot inhibited Tn4731 from transposing to it, indicating that the CTAG sequence is responsible for the preferential transposition of Tn4731.An insertion sequence (IS) is a discrete DNA segment which can transpose from one site to another in bacterial plasmids, chromosomes, and phages (for a recent review, see reference 5). A DNA segment which is flanked by the same IS elements can also transpose together with the IS elements as a unit called a composite transposon (Tn). Transposition of IS and Tn elements occurs at various sites with or without specificity (2,8,11,15,22,29).IS630 is a 1,153-bp element present in the Shigella sonnei chromosome in multiple copies (19,29). A composite transposon, Tn4731, which has two inverted IS630 insertion sequences that flank the sequence of the tetracycline resistance plasmid pHS1, has been shown to transpose to the dinucleotide 5'-TA-3' in the core of at least 4-bp palindromic sequences, such as CTAG, TTAA, and ATAT, in ColEl in Escherichia coli (29). There are two hot spots for transposition of Tn4731 within each of the inverted repeat sequences of 13 bp in a rho-dependent transcription terminator located downstream of the cea gene in ColEl (29).To clarify the determinants for the site-specific transposition of Tn4731, we have studied transposition of Tn4731 to pUC118 and its derivatives. We report here that Tn4731 can transpose also to TA sequences at four sites in pUC118 and its derivatives. We also show that the preferential transposition is determined by the CTAG sequence containing the target site, but not by the sequences surrounding the CTAG sequence. The two transpositional hot spots previously identified in ColEl by Tenzen et al. (29) are actu...

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Altered DNA conformations detected by mung bean nuclease occur in promoter and terminator regions of supercoiled pBR322 DNA

Cited by 81 publications

References 68 publications

Single-stranded-DNA-binding protein-dependent DNA unwinding of the yeast ARS1 region.

Single-stranded-DNA-binding protein-dependent DNA unwinding of the yeast ARS1 region.

Analysis of Streptococcus pneumoniae sequences cloned into Escherichia coli: effect of promoter strength and transcription terminators

Preferential transposition of an IS630-associated composite transposon to TA in the 5'-CTAG-3' sequence

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